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  • Review Article
  • OPEN ACCESS

Application and Progress of Cultured Models of Gallbladder Carcinoma

  • Jiali Xing1,# ,
  • Peiwen Ding2,3,#,
  • Xueshuai Wan1 ,
  • Gang Xu4 ,
  • Yilei Mao1 ,
  • Xinting Sang1,
  • Shunda Du1,*  and
  • Huayu Yang1,* 
 Author information  Cite
Journal of Clinical and Translational Hepatology   2023;11(3):695-704

doi: 10.14218/JCTH.2022.00351

Abstract

Gallbladder carcinoma (GBC) is a malignant tumor of the biliary system that is aggressive, difficult to detect early, and has a low surgical resection rate and poor prognosis. Appropriate in vitro growth models are expected to focus on the study of the biological behavior and assess treatment effects. Nonetheless, cancer initiation, progression, and invasion include spatiotemporal changes and changes in the cell microenvironment intracellular communication, and intracellular molecules, making the development of in vitro growth models very challenging. Recent advances in biomaterial methods and tissue engineering, particularly advances in bioprinting procedures, have paved the way for advances in the creative phase of in vitro cancer research. To date, an increasing number of cultured models of gallbladder disease have emerged, such as two-dimensional (2D) GBC growth cell cultures, three-dimensional (3D) GBC growth cell cultures, xenograft models, and 3D bioprinting methods. These models can serve as stronger platforms, focusing on tumor growth initiation, the association with the microenvironment, angiogenesis, motility, aggression, and infiltration. Bioprinted growth models can also be used for high-throughput drug screening and validation, as well as translational opportunities for individual cancer therapy. This study focused on the exploration, progress, and significance of the development of GBC cultural models. We present our views on the shortcomings of existing models, investigate new innovations, and plan future improvements and application possibilities for cancer models.

Graphical Abstract

Keywords

Gallbladder cancer, 2D gallbladder cancer cell culture, 3D gallbladder cancer cell culture, Xenograft model, 3D bioprinting technology

Introduction

Gallbladder carcinoma (GBC) is one of the most life-threatening diseases worldwide. Its morbidity and mortality are increasing, with 70–80% of patients diagnosed at an advanced stage.1 Surgery remains the only possible treatment for GBC.1 However, because of the atypical clinical symptoms of GBC, lymphatic metastasis, and distant metastasis are prone to occur, so only 10% of patients are in a state suitable for surgical resection at the time of diagnosis.2 For patients with advanced GBC, three classes of chemotherapy drugs are available, gemcitabine, fluoropyrimidines, and platinum-based compounds. Monotherapy has limited efficacy.3 A combination of gemcitabine and cisplatin (CDDP) improves survival in such patients.4 Radiation therapy has limited efficacy in patients with advanced disease. Targeted therapy against the epidermal growth factor receptor (EGFR) inhibits proliferation in vitro and may provide an effective therapeutic modality in the future.5 However, comprehensive treatment is still ineffective and the prognosis is very poor. The 5-year survival rate is only 5–15%.6 Strengthening basic research in the occurrence and progression of GBC is the key to improve the prognosis of patients.

GBC appears to result from a combination of genetic susceptibility and environmental factors.7 A hypothetical oncogenic process is gallstone-induced inflammation → p53 mutation (↓) → K-ras mutation (↑).8 Several diseases and chronic inflammation are considered risk factors for GBC, including cholelithiasis, porcelain gallbladder, gallbladder polyps, chronic salmonella infection, and congenital bile duct cysts.9 The mechanism of cancer occurrence and development has not been fully elucidated, which seriously affects the prognosis and treatment of the disease. Little is known about the biological characteristics of GBC, mainly because a complete in vitro culture system is lacking. In vitro tumor cell culture is one of the basic conditions for tumor research and is central to many fields such as tumor occurrence, development, invasion, metastasis mechanism, early diagnosis, and treatment evaluation.

Traditional tumor models are mainly used in in vitro 2D and biological models, while emerging ones include three-dimensional (3D) spheroids and organoid models. Existing 2D models have discovered and identified tumor invasive growth, including genetic and epigenetic modifications. Nonetheless, 2D models cannot recapitulate key elements of the in vivo microenvironment. Animal models including xenografts, chemical induction, and genetic manipulation have often been used in preclinical exploration and in the study of individualized treatment of cancer.10 3D cancer spheroids are growth groups formed in vitro from 3D cell cultures. These clusters reflect the morphological structure of human tumors, and are useful for precise examination of drug efficacy.11 A tumor organoid model is a new method of tumor research emerging in recent years.12 Organoids are derived from stem cells and can recapitulate certain aspects of organ structure and function, including differentiation into multiple cell types.13

A “perfect” in vitro tumor model should (1) mirror the three-layer developmental environment of growing cells in vivo, (2) mimic the cooperation between cancer and stromal cells, (3) replicate the relevant pathological features of patients, and (4) have an adaptable and time-saving development cycle.14 It is feasible to tune various parts of the cancer microenvironment and subsequently control cellular capabilities. Given the difficulties, new developments in state-of-the-art 3D bioprinting innovations have been significantly adopted and used in cancer fields by developing dynamic biomolecular/cytokine perspective models, invasive proliferation, and cell-cell communication models.15,16 The main purpose of this review is to describe the application and progress of GBC culture models, and to provide ideas for the basic and clinical translational research of GBC (Fig. 1) .

Timeline of the development of different models of gallbladder cancer cell culture.
Fig. 1  Timeline of the development of different models of gallbladder cancer cell culture.

The number of gallbladder cancer culture models is increasing, and now includes two-dimensional gallbladder cancer cell culture, three-dimensional gallbladder cancer cell culture, xenograft models, and three-dimensional bioprinting technology. GBC, gallbladder carcinoma; 2D, two-dimensional; 3D, three-dimensional.

2D gallbladder cancer cell culture

More than a dozen GBC cell lines are currently available, including G-415, GB-d1, GBK-1, and KMG-A (as shown in Table 1).17-38 The first established GBC cell line, G415, expresses epithelial cadherin (E-cad) and forms cystic or spherical 3D structures. And a plasmid containing murine E-cad cDNA was transduced into G415 cells to stop E-cad expression.39 The E-cad-transduced cell line had a typical epithelial shape and forms multicellular cyst-like structures. Shimura et al.17 found that removal of fibroblasts from GB-d1 cell cultures resulted in a decrease of tumor cell invasiveness and that the hepatocyte growth factor (HGF) secreted by fibroblasts could induce the invasive growth of GB-d1. GBC-SD, the first domestic GBC cell line is mainly composed of polygonal, spindle, and square cells.18 SGC-996 is another domestic GBC cell line. It has a typical epithelial cell morphology, and neither the morphology nor the chromosome number change significantly in long-term culture. Liu et al.20 successfully established a new cell line, TJ-GBC2, which has a characteristic epithelial tumor morphology and phenotypes consistent with primary GBCs, such as polygonal and irregular cell shapes, CA19-9 and AFP levels were elevated, and E-cadherin was expressed. The cell line is also more invasive and the highest migratory ability compared with other GBC cell lines. Zhou et al.21 established a novel ZJU-0430 cell line from a primary GBC. It had various cell morphologies and characteristic epithelial morphologies, with a population doubling time of 19.81 h. ZJU-0430 cells have greater migration, invasiveness, and proliferative ability than GBC-SD cells in vitro. In addition to the primary GBC cell line, Wang et al.22 established the EH-GB2 cell line from primary culture of a liver metastases from a 65 year-old patient cell line. It has a doubling time of approximately 48 h in cell culture and elevated expression of vimentin, Snail, Twist, matrix metalloproteinase 1 (MMP-1), and MMP-2 mRNAs, which are characteristic of epithelial-mesenchymal transition. Another metastatic gallbladder disease cell line, EH-GB1, reported by Li et al.23 was passaged over 20 generations, had an aggressive epithelial morphology and a stable developmental pattern for 24 h. EH-GB1 cells grew in mice after subcutaneous inoculation and were positive for the cancer marker CA19-9. The cell line consistently expressed green fluorescent protein (GFP) and Red 2. In terms of pathological types of GBC, Shinichi et al.24 established a human GBC cell line TYGBK-1 from papillary tubular adenocarcinoma patients. It had p53 missense mutations, produced CA19-9, and was sensitive to gemcitabine.

Table 1

Reports of human gallbladder cancer cell lines

AuthorCell lineReport yearCharacteristicDoubling time (h)
Koyama25G-4151980Differentiated gallbladder cancer cell line–
Morgan26COLO 3461981polygonal cells36
Knuth27Mz-ChA-1.21985Secretes C3C4C5; express AFP or ferritin72
Egami28r51 GBK-11986polygonal cells; express CSF 4343
Maruiwa29KMG-A1987Expresses AFP–
Homma30NOZ1988Polymorphism; expresses CEA or ferritin; K-RAS mutation48
Johzaki31FU-GBC-11989Expresses CEA, CA199, EMA120
Tsubono32GBC-GN1992Well-differentiated adenocarcinoma; cisplatin-sensitive7
Nakano33HAG-11994Moderately differentiated adenocarcinoma of the gallbladder; no mutation or amplification of H-, K-, or N-ras genes detected26
Shimura17GB-d1,21995Secretes IL-31–
Nishida34FU-GBC-21997Signet ring cell; express CEA, CA199, EMA43
Yamada35OCUG-11997Poorly differentiated adenocarcinoma; secreting TA-447.1
Park36KMCH-11999secrete IL-639
Liu18GBC-SD2000Polymorphism, rapid proliferation, congenital drug resistance21.4
Ebinuma37PTHrP-GBK2002Expresses PTHrP 4848
Yang19SGC-9962003Typical epithelial cell morphology46
Ghosh38TGBC2004Expresses CEA, CA19-9, MUC-1, and c-erbB230–96
Li23EH-GB12010Gallbladder carcinoma liver metastases cell line, typical features of malignant epithelial cells, the tumorigenic rate of mice is 100%, and CA19-9 has strong positive expression24
Wang22EH-GB22011Gallbladder carcinoma liver metastases cell line, EMT, highly expressed Vimentin, Snail, Twist, MMP-1, and MMP-2 mRNA48
Shinichi24TYGBK-12012p53 missense mutation, showing sensitivity to gemcitabine48
Liu20TJ-GBC22017Abnormal chromosome structure and number, hypertetraploidy; coexistence of polygonal, fusiform, and irregularly shaped cells-
Zhou21ZJU-04302019Variety of cell morphologies and characteristic epithelial morphologies19.81

AFP, alpha-fetoprotein; CEA, carcinoembryonic antigen; CSF, colony-stimulating factor; EMA, epithelial membrane antigen; EMT, epithelial-mesenchymal transition; MMP, matrix metalloproteinase.

In vitro drug screening in 2D culture has the advantages of high-throughput and convenient screening, and is widely used in preclinical trials of drugs. An in vitro culture model of GBC cell lines has been successfully used in the study of the mechanism of genetic and molecular changes of GBC and drug sensitivity experiments, especially the abnormality of GBC suppressor genes, proto-oncogenes, and DNA repair genes, microsatellite instability, and epigenetic changes. Qin et al.40 used v-src- or cH-ras-transfected HAG-1 human GBC cell lines to find that activated Ras and Src induced resistance to gefitinib by activating one or both of the Akt and Ark signaling pathways. It has also been reported that KRAS, CDKN2A/B and other gene mutations exist in GBC, and are closely related to the invasiveness and metastasis.6,41 However, studies have shown that epigenetic changes and tumor suppressor gene methylation in GBC are abnormally increased and closely related to patient survival.42 Gao et al.43 examined the high expression of NSUN2 in GBC tissues and GBC-SD cell lines and found that NOP2/SunRNA methyltransferase 2 promoted GBC progression through the interaction partner RPL6. Additionally, the role of noncoding RNAs in the occurrence and development of GBC has recently received attention. In the NOZ and SGC-996 cell lines, miR-122 inhibited the proliferation of GBC by interfering with the expression of PKM2, MALAT1 promoted the proliferation and metastasis of GBC by activating the ERK/MAPK pathway.44 The GBC cell line has a high xenograft tumor formation rate, which is also an important criterion in the identification of cell lines. Tsubono et al.32 used the well-differentiated cell line GBC-GN to establish a nude mouse tumor-bearing model to test the antitumor anticancer effects drugs such as CDDP and 5-fluorouracil (5-FU), alone or in combination. Only CDDP was effective in GBC-GN model, and CDDP+5-FU had an inhibitory effect on both malignant tumors.

However, in no case can 2D models retain the key qualities of the in vivo microenvironment. Some studies have suggested cancer cells derived from patients under 2D conditions experience a reduction or complete loss of certain receptors and marker molecules.45 Comparison of 2D and 3D models shows huge differences in phenotype, gene and protein expression, and drug sensitivity.46 These elements make 2D an unacceptable technique for accurate drug screening. Due to factors such as genetic drift, loss of heterogeneity, and cell line contamination, the experimental outcomes of cell lines are not very robust in the final animal experiments. That may be because the cell culture process in a 2D environment in vitro cannot truly reflect the interactions of the tumor microenvironment surrounding the tumor cells in vivo. Primary tumor cells can better retain the characteristics of host cells, but the current in vitro culture of primary tumor cells is limited due to difficulties in cell culture technology and cycle. Compared with the widespread application of GBC cell lines, the culture of primary GBC cells has also been further developed and applied. Bo et al.47 collected resected lesions from patients with gallbladder adenoma and GBC, and successfully cultured primary gallbladder tumor cells in vitro. To improve the efficiency of primary cell culture, Noggin, epidermal growth factor (EGF), fibroblast growth factor 10 (FGF10), and insulin-like growth factor (IGF) were added to the cell culture medium, and Wnt3a, Rspo1, and prostaglandin E2 were added to the gallbladder adenoma medium at the same time. The researchers then successfully established organoids from primary GBC cells, which were expanded for at least 10 passages and stably cultured for more than 3 months. Feng et al.48 successfully constructed 38 patient-derived primary cancer cell lines with multiregional sampling of tissue samples from seven patients with GBC surgery, and studied the heterogeneity of GBC. In addition to the culture of primary GBC cells, Chen et al.49 cultured primary GBC-related fibroblasts, using enzymatic digestion and tissue blocks for the primary culture of human GBC-related fibroblasts and purification by differential adherence.

Recent progress of the in vitro culture of primary tumor cells is reflected by the improvement of cell culture media, cell-separation methods, and conditional reprogramming of cultured cells.50–53 In terms of the tumor cell culture model, a 3D culture model is gradually replacing 2D culture model.

3D gallbladder cancer cell culture

In recent decades, various methods for culturing 3D spheroids have been developed. 3D culture methods can be divided into scaffold-containing and scaffold-free cell culture methods. Scaffolding materials can be divided into natural scaffolds using matrigel, collagen, gelatin, laminin, chitosan, hyaluronic acid, and synthetic scaffold materials by polyester degradable polymer.54–57 Scaffold-free culture models mainly refer to the application of various physical methods to prevent tumor cells from adhering to the wall, to suspend tumor cells in the medium, and to promote cell aggregation and growth to form tumor cell spheres. It includes ultra-low adhesion culture plates, dynamic rotation culture, hanging drop methods, and microfluidic chips and acoustic manipulation, which have appeared in recent years.58–62

The 3D cell simulates the in vivo microenvironment of tumor cell growth. Under 3D culture conditions, almost 100% of the surface area of the cells is in contact with other cells or substrates, and the cultured cells have characteristic biological signal transduction.63,64 In the 3D culture environment, cells have a scattered distribution in clusters, forming homogeneous scattered microcell spheres. In the microspheres, there may be cell junctions and paracrine communication that cannot be realized in 2D culture, and act to maintain the tumor characteristics.46

Currently, 3D culture of GBC uses 3D gels. Wan et al.39 systematically compared the cell phenotype, morphogenesis, and ultrastructure of G-415 GBC cells containing an expression plasmid of mouse E-cad cDNA in 2D and 3D gel culture conditions. In 3D gels, the GBC cells formed round multicellular cyst-like structures that secreted mucous material from the apical surface into the cyst cavity and had the potential for hyperproliferation, which was in contrast to 2D culture.

The main applications of 3D culture model are in drug sensitivity studies and the mechanism of the GBC microenvironment. Jiang et al.65 established a 3D cell culture model of GBC using preparations of cell-containing 3D collagen to study the killing effectiveness and mechanism of a novel nanodrug delivery system in GBC cells. Experiments found that the interaction between cells in 3D culture was significantly enhanced, and the biological characteristics of multicellular spheres were mainly characterized by cell cycle arrest and early apoptosis reduction, which may have been related to multicellular drug resistance. In terms of the occurrence mechanism of GBC, cancer-associated fibroblasts (CAFs) and vasculogenic mimicry (VM) have important roles in the malignant growth of GBC. Pan et al.66 used a 3D collagen gel co-culture of human GBC cells and fibroblasts or HUVECs, which showed that CAFs upregulated NOX4 expression to promote VM formation and cancer development, possibly by paracrine secretion of IL6 to complete the IL-6-JAK-STAT3 signaling pathway. NOX4 is a critical gene for VM development in GBC. It is aberrantly expressed in the cancer cells and stroma and is associated with poor prognosis in GBC patients.

New 3D culture methods incorporating acoustic manipulation and microfluidics that avoid limitations of the use of other 3D culture methods for GBC, have been used in studies of bladder cancer, breast cancer, and other malignant tumors (Fig. 2). Gong et al.61 printed tumor organoids with acoustic droplets that mimicked the immune microenvironment of bladder tumors. Their platform facilitated rapid formation of tumor organoids that preserved the original tumor immune microenvironment and the establishment of a personalized bladder cancer immunotherapy model. Using a droplet microfluidic platform, Chen et al.67 prepared delicate structures with fibroblasts in the outer layer and hepatoma cells in the inner layer and achieved homogenization to generate heterogeneous pseudospheres that were used to study interactions between tumors and stromal cells.

Different types of Three-dimensional culture models include scaffold-containing and scaffold-free cell methods.
Fig. 2  Different types of Three-dimensional culture models include scaffold-containing and scaffold-free cell methods.

(A) Three-dimensional gel cell culture model; (B) Low-adhesion cell culture; (C) Dynamic spin cell culture; (D) Acoustic control of cell culture; (E) Suspension cell culture; (F) microfluidic cell culture; (G) 3D bioprinting cell culture.

However, 3D culture methods have downsides. Scaffold-based 3D cell culture involves conventional or mixed polymers as the extracellular matrix (ECM), thereby supporting cell development. Commonly used techniques include matrix coverage/embedding culture and microcarriers. Robust cell-ECM interactions can understood by the appearance of ECM material. Nonetheless, the outer materials are toxic and antigenic, separating the spheres from the platform material is difficult, and the platform materials may affect drug diffusion and responses, which results in low reproducibility of drug testing results.68 Frameless 3D cell culture does not require suspended external materials or power. Rotating and spinner flasks, hanging drops, ultra-low attachment plates, and microporous plates are devices that are routinely used in 3D culture. Rotating and spinner flasks can create stress that is strong enough to damage cells during culture, which makes it challenging to control the consistency of spheroids. Hanging drop assays require intensive work and long-term culture. They are unstable, due to the evaporation of media from the droplets. Ultra-low linker plates have low efficacy, and there is only one spheroid in a culture well, making it difficult to control spheroid uniformity. Microporous plates use specially created microwells through micropatterning. To keep up with suspending cells, the well surface should be covered to reduce attachment, which may hinder the effects of drug molecules.69

Gallbladder cancer organoids

Organoids are a novel 3D cell culture technology that can grow primary tissue plants or single cells into self-organizing tissues. Organoids can retain histological features, expression profiles, unique markers, and many other features of the original tissue.70 The extraordinary strength of this model is that organoids can be cultured over a long period of time (especially for patient-specific tumor cells) and passaged in 3D over a long time. Additionally, organoids can preserve the histological, immunohistochemical, and genetic heterogeneity of the original tumor, making them suitable for high-throughput drug screening.

Lugli et al.71 isolated gallbladders from 2 month-old mice, collected them and incubated them with PBS/EDTA for 2 h. Small clusters of cells were isolated and implanted into matrigel. The cells were incubated in a medium containing nicotinamide and various growth factors including EGF, fibroblast growth factor 10, and noggin. The gallbladder organoids that developed reproduced stably for more than a year and expressed the stem cell marker Prom1 and the transcription factor Sox17.

GBC organoids have been successfully used to explore the mechanism of occurrence and development of GBC and drug sensitivity (Fig. 3). One study obtained GBC organoids from mice in which the TP53 gene was inactivated and cultured them after infection with salmonella.72 Organoids with a history of infection lost cohesion and polarity, had enlarged nuclei, irregular and prominent nucleoli, and exhibited neoplastic transformation. Organoids without infection had normal epithelial tissue. Therefore, salmonella has a susceptible genetic background and can promote cellular tumorigenesis. The current first-line chemotherapy regimen for GBC, gemcitabine combined with CDDP, has a median survival of less than 1 year.73 García et al.74 found that Yes-associated protein 1(YAP1) was highly expressed in 60% of patients with chronic cholecystitis and advanced GBC. High nuclear expression of YAP1 was associated with poor overall survival in patients with subserous GBC. They also demonstrated that inhibition of YAP1 by verteporfin (VP) reduced migration and invasion of GBC cell lines. To overcome the lack of an intact immune system, Shingo et al.75 recently established a new model using lentiviral Cre transduction and CRISPR/Cas9 gene editing to generate mutant Kras and Trp53-loss gallbladder organoids in vitro. Sections of organoid-defined subcutaneous tissue that were sutured to the outer surface of the gallbladder in syngeneic mice produced carcinomas in situ that resembled human GBCs histology and gene transcription. The model revealed the penetration of similar immune cell subsets in subcutaneous and orthotopic tumors, confirming a suitable immune environment during carcinogenesis, demonstrated the in vivo efficacy of gemcitabine.

Applications and steps of three-dimensional gallbladder cancer cell organoids.
Fig. 3  Applications and steps of three-dimensional gallbladder cancer cell organoids.

Gallbladder cancer organoids can be constructed by three-dimensional culture of tumor cells isolated from patients or mice and have been successfully used to explore the mechanism of occurrence and development of gallbladder cancer and studies of drug sensitivity. 3D, three-dimensional.

There are many limitations of the use of GBC organoid technology. First, many complex and expensive cytokines need to be added to maintain the tumor microenvironment of the organoid. Second, the success rate of in vitro tumor organoid construction depends on the tumor’s own proliferative capacity. GBC and other low-proliferation tumors, have a low success rate in constructing organoids in vitro, which makes the use of tumor organoid technology less extensive than that of patient-derived xenograft (PDX) models.76 Third, its deficiency in interstitial cell components and the absence of cell-to-cell interactions make it impossible to reconstruct the tumor microenvironment.

Gallbladder cancer xenograft models

Human tumor xenograft models involve transplantation of human primary tumor tissue into immunodeficient animals. At present, there are two types of xenograft models of GBC, subcutaneous and orthotopic (Fig. 4).77 Subcutaneous tumor models can be prepared by the cell suspension injection or tissue block methods. A cell suspension can be injected subcutaneously or injected intraperitoneally into the lateral femoral side of nude mice. Subcutaneous tumors that have grown into blocks with a diameter of 1.0 cm can be removed and cut into pieces of about 1 mm in size and reinoculated. Subcutaneous tumors prepared by the cell suspension method can be inoculated under the serosa of the gallbladder by surgery.

Classification and steps of preparing gallbladder cancer cell xenograft models.
Fig. 4  Classification and steps of preparing gallbladder cancer cell xenograft models.

Xenograft models of gallbladder cancer include subcutaneous and orthotopic tumor models. Subcutaneous tumor models can be constructed from injection of cell suspensions or tissue blocks. The injection is either subcutaneous or intraperitoneal into the lateral femoral side of nude mice.

Patient-derived xenografts propagated in immune-compromised mice at ectopic or orthotopic sites replicate the pathological features of patients to a certain extent. Compared with the cell transplantation model (i.e. cell-derived xenograft, CDX) that directly transplants the cell line into nude mice because the PDX model comes directly from the patient, it will not lose heterogeneity and undergo genetic drift like the cell line, so it can better maintain the original tumor morphology, metastasis characteristics, mutation spectrum, and drug response.69

Xenograft models are widely used in drug screening and individualized treatment of various tumors, such as melanoma,78 non-small cell lung cancer,79 breast cancer,80 ovarian cancer,81 and hepatocellular carcinoma.82 Many studies using subcutaneous transplantation models have been carried out by researchers after the successful cultivation of cell lines, and have mainly focused on anticancer drugs and early diagnosis of tumors. Pan et al.83 used the NOZ cell line to establish a nude mouse subcutaneous tumor and GBC orthotopic models in nude mice, in which they analyzed and compared the mechanisms of GBC metastasis. The nude mice in the orthotopic tumor transplant group showed signs of spontaneous metastasis. The tumors showed strong invasiveness, but there was no obvious spontaneous metastasis occurred in the subcutaneous tumor group. The results reflect the relationship of the invasiveness of tumor cells to the surrounding environment of the organ. In a study of the antitumor effectiveness of gemcitabine, Mita et al.84 also inoculated NOZ cells into the gallbladder, founding that lymph nodes, liver, and lung metastasis occurred in nude mice. To determine whether the trauma caused by the puncture of the abdominal wall could promote the implantation of tumors at the puncture site, some researchers injected the GBC cell line GB-d1,2 cells into the abdominal cavity of nude mice undergoing different surgical operations. They found that most of the patients with peritoneal injury, laparoscopic operation, peritoneal burns, or liver-surface burns experienced tumor implantation and metastasis at the operation site, and that implantation was greatly reduced by repairing the peritoneum.85 Animal-induced models of GBC were reported as early as 1974.86 Some people believe that azotoluidine nitrosamines, artificial cholesterol stones, and other materials implanted in the gallbladder, can lead to the occurrence of GBC, but successful cases have been very rare. Talima et al.87 successfully established a GBC model in female Syrian mice. First, distal common bile duct ligation and cholecystoduodenal anastomosis were performed, and nitrosamine feeding was administered 4 weeks after the operation. The GBC showed papillary growth, and a morphology similar to that of human GBC. Bromodeoxyuridine and anti-bromodeoxyuridine monoclonal antibody immunohistochemical staining confirmed that gallbladder duodenal anastomosis significantly accelerated the activity of gallbladder epithelial cells, which may play a major role in the carcinogenesis of gallbladder epithelial cells. Reflux and stasis of pancreatic juice can lead to chronic inflammation of the gallbladder, intestinal metaplasia, and finally to the occurrence of differentiated GBC. A study by Ikemaltsu et al.88 confirmed the above perspective, and found that nitrosamines induced GBC in mice, and oral administration of cholestyramine inhibited carcinogenesis. It was inferred that bile acid is involved in gallbladder carcinogenesis.

A major limitation of xenograft models is the loss of human tumor stroma, which is completely replaced by murine stroma in the second generation.89 To overcome it, co-implantation of human stromal cells with primary tumor tissue optimizes traditional xenograft models. In addition, the long cycle of xenograft model establishment limits its application in guiding the treatment of the original patient. The time gap was bridged by the rapid in vivo drug sensitivity assay of mini-xenograft model, which can test the response to antitumor drugs within 7 days.90 To establish a mini-xenograft model, cancer cell suspensions were transferred to a modified microcapsule and hollow-fiber culture system. The capsules were subcutaneously transplanted into immunodeficient mice, and the mice were subsequently treated with antitumor drugs. Treatment response was assessed by cell proliferation. However, xenograft models established in immunocompromised mice are ineffective in studying the tumor microenvironment, including the infiltration of immune cells and crosstalk between the tumor and the immune system. To overcome those challenges, several humanized mice have recently been developed, including genetically engineered humanized mice and immune humanized mice.91

Emergence of 3D bioprinting technology

3D bioprinting is essentially the same as organoids. and it is also an in vitro 3D culture technology based on matrigel, but with the help of precision engineering instruments, precise models can be constructed with millimeter-level errors. 3D printing is an innovative process using rapid prototyping materials, layer-by-layer superposition, and point-by-point printing to produce 3D graphics. This rapid and lowest-cost innovation has some control over the development of multicellular, multimaterial, and multibiomolecular structures in a multilevel space. It can also reproduce the growth microenvironment and mimic the complex organic structure of cancer tissue.92 Additionally, different cell components can be added. The multichannel printing has the advantages of controlling the 3D position of each cell component, restoring the different distribution characteristics of different cells in the tissue and achieving the purpose of accurately simulating the in vivo microenvironment. That is sufficient to overcome the randomness of organoid technology and the defects of homogenization of cell distribution.93

The use of 3D bioprinting has been explored in personalized treatment schemes and construction of a model tumor microenvironment consistent with the in vivo parental tumor tissue with respect to gene sequences, gene expression levels. We have used 3D bioprinting technology to successfully construct human normal liver tissue,94 liver cancer tissue,95 and intrahepatic cholangiocarcinoma tissue,96 confirming that 3D printing has the advantages of a high success rate and stability for study of drugs and tumor pathogenesis (Fig. 2G). Recently, our experimental team used 3D bioprinting to construct an in vitro multicellular GBC model that accurately simulated the tumor microenvironment of GBC and can be used to explore the mechanism of occurrence and development of GBC and to conduct drug sensitivity experiments.

3D biological printing has disadvantages compared with 3D culture. Whether it is an ordinary mechanical extrusion printer or a newer type such as light curing printer, building a specific model of cells requires more preliminary work, such as the preparation of bigels, and takes a long time for completion. Long-term exposure of cells to biological ink leaving the culture medium also affects their function.14 Additionally, the mechanical compression of the extrusion printer can also cause cell damage that can result in the death of some fragile primary cells soon after printing, making it impossible to successfully build a model. Further development of biomaterials and mechanical engineering technology is needed.14

Outlook

Although GBC still faces many problems such as a high degree of malignancy, a single treatment option, and a low 5-year survival rate, they will gradually be solved with the in-depth understanding of the pathogenesis, clinical diagnosis, and treatment. By comparing the advantages and shortcoming of above models, it contributes to provide the future directions (as shown in Table 2). With the continuous updating of primary tumor cell culture technology, long-term in vitro culture of primary GBC cells obtained from patients will facilitate the realization of individualized and precise treatment. In view of the increasingly important role of 3D tumor cell culture, additional models will be used to explore the mechanism of GBC occurrence and development in the future. 3D bioprinting techniques have become the focus of current research because they can model cell-to-cell interactions. More importantly, 3D bioprinting technology can print multiple cells to simulate the tumor microenvironment and better recapitulate the complexity of tumors, which is of great significance for clinical diagnosis and personalized medicine of tumors. Currently, research on 3D bioprint culture models of GBC are in their infancy. The construction success rate, application, and prospects need more research. However, that does not affect the research value and bright prospects of 3D bioprinting technology for GBC.

Table 2

Comparison of common in vitro gallbladder cancer culture models

Feature2D cell line3D cell cultureOrganoidsXenograft model3D bioprinting
MethodCell culture dishCollagen-based scaffoldThermosensitive basement membrane extract; hydrosolImplantedBioink;3D printer
In vivo/vitroVitroVitroVitroVivoVitro
Success rateHighHighMediumLowHigh
AdvantagesLow maintenance cost; high reproducibility; simple and feasible operation; real-time assessment of cell survival and proliferationEasy to build; high reproducibilityExplore the histological and biological characteristics of the original tumor; simulate the complex tumor microenvironmentStudy tumor biological interactions in vivo; simulate tumor microenvironment; long-term culture;Retain the biological and genetic characteristics of the original tumor; simple and convenient operation; long-term survival and passage
DefectsEasy to accumulate mutations, tumor cells lack interaction with other surrounding cellsLack of histological and morphological featuresHighly heterogeneous; depending on the inherent proliferation ability of the original tumorTumor environment different from the human body; interference of animal’s own immune system; high cost of facilities and skill training; ethical controversyExpensive instruments; mechanical scissors during printing
Number of papers published778214238–

2D, two-dimensional; 3D, three-dimensional.

In addition to GBC and malignant tumors, current research in the causes, occurrence, development, and treatment of benign diseases of the gallbladder, such as gallbladder stones, adenomyosis of the gallbladder, and others are mainly carried out in animal models and mathematical exercises.97,98 The main experimental animal models of gallstones are implanting human gallstones into animal gallbladders and observing the changes and the influence of Chinese and Western medicines, the other models are induced by food.98 The former is suitable for the experimental study of promoting gallstones and expelling stones but not for research on the cause of gallstones and stone prevention. The latter can be used to study stone prevention, but has a long observation time of at least 2 months. The use of in vitro culture models for the study of benign gallbladder disease is currently very limited. More models of gallbladder disease are expected to appear in the future.

Abbreviations

2D: 

two-dimensional

3D: 

three-dimensional

5-FU: 

5-fluorouracil

AFP: 

alpha-fetoprotein

CAF: 

cancer-associated fibroblasts

CDDP: 

cisplatin

CDX: 

cell-derived xenograft

CEA: 

carcinoembryonic antigen

CSF: 

colony-stimulating factor

CT: 

computed tomography

E-cad: 

epithelial cadherin

ECM: 

extracellular matrix

EGF: 

epidermal growth factor

EGFR: 

epidermal growth factor receptor

EMT: 

epithelial-mesenchymal transition

FGF: 

fibroblast growth factor

GBC: 

gallbladder carcinoma

GFP: 

green fluorescent protein

HGF: 

hepatocyte growth factor

IGF: 

insulin-like growth factor

JSH: 

Japan Society of Hepatology

LCSGJ: 

Liver Cancer Study Group of Japan

MMP: 

matrix metalloproteinase 1

MRI: 

magnetic resonance imaging

PDX: 

patient-derived xenograft

TACE: 

transarterial chemoembolization

VM: 

vasculogenic mimicry

VP: 

verteporfin

YAP1: 

yes-associated protein 1

Declarations

Funding

This work was supported by grants from the National Natural Science Foundation of China (81972698).

Conflict of interest

The authors have no conflict of interest related to this publication.

Authors’ contributions

Study conception and design (JX, GX, XW), drafted the first version of the manuscript (JX, GX, XW, YM, XS), edited and revised the manuscript (PD, SD, HY). All authors have approved the final version of the article, including the authorship list.

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Application and Progress of Cultured Models of Gallbladder Carcinoma

Jiali Xing, Peiwen Ding, Xueshuai Wan, Gang Xu, Yilei Mao, Xinting Sang, Shunda Du, Huayu Yang
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